This assay was carried out as described in a previous report.(30 (link)) CD24 (clone SN3b) antibody was purchased from Thermo Fisher Scientific (Cheshire, UK). HIF-1α (NB100-105) and HIF-2α (NB100-122) were purchased from Novus Biologicals. HISTOFINE SAB-PO (M and R) kit (Nichirei, Tokyo, Japan) and 3,3′-diaminobenzidine (DAB, Muto Pure Chemicals, Tokyo, Japan) were used for the signal detection and amplifications.
Hif 2α nb100 122
Manufactured by Novus Biologicals
Sourced in United States, Mongolia
HIF-2α (NB100-122) is a protein-specific antibody that recognizes the hypoxia-inducible factor 2-alpha (HIF-2α) protein. HIF-2α is a transcription factor that regulates the expression of genes involved in cellular responses to hypoxic conditions.
Automatically generated - may contain errors
Lab products found in correlation
Hif 1α, by BD (2 mentions)
Actin hrp, by Merck Group (2 mentions)
Hpa001849, by Merck Group (2 mentions)
Riok3, by Abnova (2 mentions)
Ca9 clone m75, by Abcam (2 mentions)
Tmod3, by Merck Group (2 mentions)
Ab113692, by Abcam (2 mentions)
Hif 1α nb100 105, by Novus Biologicals (1 mentions)
Phospho p70 s6 kinase thr389, by Cell Signaling Technology (1 mentions)
P70 s6 kinase, by Cell Signaling Technology (1 mentions)
Hif 1α, by GeneTex (1 mentions)
Hrp conjugated anti rabbit or anti mouse antibody, by Cell Signaling Technology (1 mentions)
Vinculin, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Gtx30114, by GeneTex (1 mentions)
Ab40800, by Abcam (1 mentions)
Ab107257, by Abcam (1 mentions)
Hif 1α nb100 479, by Novus Biologicals (1 mentions)
Hif 1α gtx127309, by GeneTex (1 mentions)
Nb200 598, by Novus Biologicals (1 mentions)
8 protocols using hif 2α nb100 122
1
Immunohistochemical Analysis of HIF-1α and HIF-2α
2
Immunoblot Analysis of Cellular Proteins
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Cell sample harvesting and immunoblot analysis were performed as presented previously [50 (link)]. To detect proteins, the primary antibodies β-actin (sc-47778, Santa Cruz Biotechnology, USA), vinculin (Sigma-Aldrich), HIF-1α (GTX127309, Genetex, USA), HIF-2α (NB100–122, Novus Biologicals, USA or GTX30114, Genetex, USA), p70 s6 kinase and phospho-p70 s6 kinase (Thr 389) (9202 and 9205 respectively, both from Cell Signaling, USA), and MF20 (a hybridoma producing both antibody detecting α and β MHC isoforms; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) were used in 5% non-fat milk/TBS-T at 4°C overnight. Corresponding secondary HRP-conjugated anti-rabbit or anti-mouse antibodies (in 5% non-fat milk/TBS-T, 1 h, RT; Cell Signaling, USA) were employed. Immunoreactive bands were detected using ECL detection reagent kit (Pierce, USA) and exposed to radiographic film (AGFA, Belgium). The adjustment of brightness and contrast was applied to the whole image.
3
Western Blot Analysis of Hypoxia Markers
4
Western Blot Analysis of Hypoxia Markers
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Western immunoblotting was performed as reported.21 (link) Primary antibodies used were: RIOK3 (H00008780-M01, Abnova), HIF1α (610959, BD Transduction Laboratories), HIF2α (NB100-122; Novus), CA9 (clone M75, gift of J. Pastorek, Bratislava), TPM3 (ab113692, Abcam), TPM3 (HPA009066, Sigma-Aldrich), TMOD3 (HPA001849, Sigma-Aldrich) and Actin-HRP (A3854; Sigma-Aldrich). Band densitometry was performed using the Analyze Gels tool in ImageJ (http://imagej.nih.gov/ij , version 1.47q).
5
Antibody Characterization for Western Blot Analysis
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Antibodies used for western blot analysis are as follows: HIF-1α (GTX127309) from GeneTex; HIF-1α (NB100-479) and HIF-2α (NB100-122) from Novus Biologicals; HIF-2α (#7096), pVHL (#2738), FASN (#3180) and ubiquitin (#3936) from Cell Signaling; Elongin C (sc-135895), CUL2 (sc-166506), ARNT (sc-5580), SREBP1 (sc-366), and normal mouse and rabbit IgG (sc-2025 and sc-2027 respectively) from Santa Cruz Biotechnology; CAD (ab40800), RNA polymerase II large subunit CTD (ab5408), and CA9 (ab107257) from Abcam; β-actin (A5441) and anti-Flag M2 (F1804) from Sigma-Aldrich; p65 (610869) from Becton Dickinson (BD) and REDD1 (A500-001A) from Bethyl Laboratories. For immunofluorescence, antibodies against pVHL (BD, 556347) or FASN (#3180) were used. For immunoprecipitation, antibodies against pVHL (BD, 556347), FASN (Santa Cruz Biotechnology, sc-55580) or CAD (Abcam, ab40800) were used.
6
Hypoxia-induced Chromatin Immunoprecipitation
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Cells were exposed to 20% or 1% O2 for 24 or 72 h, cross-linked with 1% formaldehyde for 20 min at 37°C, and quenched in 0.125 M glycine. DNA immunoprecipitated from the sonicated cell lysates was quantified by SYBR Green Real-time PCR (Bio-Rad) [41 (link)]. The following antibodies were used: V5 (66007-1-lg, Proteintech), HIF-1α (sc-10790, Santa Cruz), HIF-1β (NB100-124, Novus Biologicals), HIF-2α (NB100-122, Novus Biologicals), and RNA polymerase II (pSer5) (NB200-598, Novus Biologicals). PCR primer sequences are shown in Table 1 .
7
HIF-2α Expression Analysis in Cell Lysates
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Cells were lysed in RIPA buffer containing 1% NP-40, 150mM NaCl, 50mM TRIS-Cl (pH 8.0), and 1% SDS supplemented with cOmplete, EDTA-free protease inhibitor (11873580001, Roche) for 15 minutes at 4°C in a Bioruptor. Cell lysates were centrifuged at max speed at 4°C for 10 minutes, and the membrane pellets were discarded. Protein concentration was determined by bicinchroninic acid assay (BCA) protein assay (#23227, Life Technologies), after which equal amounts of protein were loaded and separated by polyacrylamide gel electrophoresis. The following antibodies were used: HIF-2α (NB100-122, Novus) and GAPDH (10R-G109a, Fitzgerald Industries).
8
Western Blot Analysis of Protein Markers
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Western blot analyses were performed as previously described48 . Primary antibodies used against Ki-67 (ab15580, 1:1000 dilution) and PEPT1 (ab55936, 1:200 dilution) were purchased from Abcam (Cambridge, Great Britain); p21 (sc-397, 1:200 dilution) from Santa Cruz Biotechnology (Texas, United States); ABCB6 (600-401-945, 1:500 dilution) from Rockland (Limerick, Ireland); ABCG2 (BXP-21, 1:200 dilution) from Convance Research Products (Emeryville, Canada); HIF-1α (AF1935, 1:400 dilution) from R&D Systems (Minneapolis, Mongolia); HIF-2α (NB100-122, 1:500 dilution) from Novus Biologicals (Littleton, United States); actin (691001, 1:500 dilution) from MP Biomedicals (Santa Ana, United States). For the second antibody, we used anti-mouse and anti-rabbit IgG HRP-conjugated antibody (1:3000 dilution) from Cell Signaling Technology (Beverly, United States).
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